The long-term goal of this project is to understand the cellular and molecular mechanisms that regulate mucociliary clearance and their dysfunction in airway diseases. The recent discovery of hyaluronan at the apex of airway epithelial cells and its regulation of ciliary function makes hyaluronan only the second known endogenously produced regulator, in addition to ATP, of human ciliary beat frequency (CBF) at the luminal side of the airway. Thus, hyaluronan must play an important role in host defense. Preliminary data suggest that hyaluronan is made by the epithelium in a form that requires degradation to stimulate CBF. Hyaluronan can be degraded in the airways by reactive oxygen/nitrogen species. Thus, during times of airway insults, hyaluronan is expected to depolymerize and start signaling via RHAMM (receptor for hyaluronic acid mediated motility or CD 168) at the apex of ciliated cells through an unknown pathway to stimulate CBF. RHAMM signaling in other systems has been proposed to work through hyaluronan-mediated association with growth factor receptors. RON, a receptor of the HGF family that can regulate CBF, is expressed at the apex of human airway epithelial cells and could mediate hyaluronan's stimulation of CBF. Thus, this proposal will test the overall hypothesis that hyaluronan is assembled in the apical membrane of the superficial epithelium where it is immobilized. Upon hyaluronan depolymerization by oxidants, signaling through RHAMM stimulates CBF and in turn mucociliary clearance. The specific aims are: 1) to test the hypothesis that hyaluronan retention at the surface occurs either by attachment to its synthase at the epithelial apex or by interaction with RHAMM; 2) to test the hypothesis that only mid to low molecular sized molecules of hyaluronan stimulate CBF; 3) to test the hypothesis that in situ depolymerization of endogenous hyaluronan by oxidants increases CBF and mucus transport rates; and 4) to test the hypothesis that signaling via RHAMM increases CBF and mucus transport rates by hyaluronan-mediated association of RHAMM with a growth factor receptor, possibly RON. The results of these experiments will provide new mechanistic insights into our understanding of airway biology as it pertains to hyaluronan, ciliary beating, mucociliary clearance, and their interactions in the airways, and could identify novel therapeutic targets for mucociliary dysfunction.